Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Protein Structure & Function > LT3 NMR Side-Chain Assignment > Flashcards

LT3 NMR Side-Chain Assignment Flashcards

1
Q

What don’t we know after doing HNCACB and CBCAA(CO)NH?

A

We know the correct order of the residues

But don’t know which amino acid the peak is yet

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What does TOCSY stand for

A

Total Correlation Spectroscopy
For ALIPHATIC side chain assignment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is TOCSY used for?

A

Correlates amide groups (N-15 labelled) to all aliphatic H (protons) in the SAME amino acid residue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What will each amide strip of a TOCSY show?

A

Each amide strop will show H chemical shift of Ha, Hb, Hy from the same residue in the 3D

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

In a TOCSY, can we always see all aliphatic protons?

A

No

If transfer is not enough, sometimes only Ha and Hb can be observed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are the axes of the TOCSY?

A

H1 vs NH

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How do we get secondary structure from chemical shift index?

A

Secondary shift = different between H/C chemical shift for each residue and that reported for the same residue type in a “random coil” conformation

Experimental - random coil = secondary

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What does the Chemical Shift Index (CSI) tell us?

A

Correlates between chemical shift tendencies and secondary structure

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are the values of a Ha or Cb secondary shift?

A

-ve for alpha helix
+ve for beta-sheet

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the values of a Ca secondary shift?

A

+ve for alpha helix
-ve for beta-sheet

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What does NOE stand for and what is it?

A

Nuclear Overhauser Effect = exist only between two H separated by less than 5 angstrom

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What does an observable NOE tell us?

A

Means the two H are in close vicinity to each other

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Describe the difference in NOE for the secondary structures

A

Alpha-helix = characterized by the close approach between residue i and i+3

Extended beta-sheet = excludes medium range distances
Close proximity of 2 strands brings about inter-strand distances

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is the difference between N-15 TOCSY and NOESY?

A

TOCSy will only protons on that SAME residue

NOESY will only show protons within 5 angstrom = including side chains from other residues

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What does intensity on a NOESY tell us?

A

How close the H are to each other

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is NOE cross-peak intensity proportional to?

A

1/r^6

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Why do HSQC-TOCSY and HSQC-NOESY show different things?

A

TOCSy = through bond expt, gives chemical shift of all H within same residues as the HN

NOESY = through space expt, gives chemical shift of all H within a distance of 5A from particular HN (amide proton)

18
Q

What do the different dxy(i, i+z), dNN(i,i+1), daN, dbN show?***

A
19
Q

Define intra-residue NOE

A

Between H within the same residue

20
Q

Name the 3 inter-residue NOE

A

Sequential = adjacent residues
Medium range = i to i+4
Long range = more than 4 residues apart

21
Q

Definer inter-molecular NOE

A

Between two molecules = dimer or beta-strands

22
Q

What NOEs define what structures of proteins?

A

Secondary = short and medium range
Tertiary = long range
Quaternary = inter-molecular

23
Q

Are daN and dNa different in NOE?

A

YES

Depict different places between residues

24
Q

What NOEs can we find in alpha-helix?

A

Sequential and medium range

dNN(i, i+1), daN(i, i+3), daN (i, i+4) and dbN(i, i+1)

25
Q

What NOEs can we find in beta-sheet?

A

Sequential and inter-strand

daN (i, i+1)

26
Q

What type of NOE defines tertiary structures?

A

Long-range NOEs

27
Q

How can we tell if beta-sheets are parallel or anti-parallel?

A

Parallel = N and N terminals will be close and C-C terminals will be close

Antiparallel = N-terminus close to C-terminus

28
Q

Are anti- or parallel beta-sheets closer and why?

A

Antiparallel sheets have closer beta-strands because the H bonds are more linear

29
Q

What is the coupling constant between?

A

J = measured between pairs of protons separated by 3 covalent bonds

Contains information about intervening torsion angle

30
Q

Which dihedral angle is used for J and why?

A

Phi angle

Because related to both secondary and tertiary structure

31
Q

What is the coupling constant for alpha-helices and beta-sheets?

A

a-helices = 4Hz
b-sheets = 9Hz

32
Q

What are the phi angles for right-handed a-helix and anti-parallel b-sheet?

A

a-helix = -57
b-sheet = -139

Larger angle = larger distance in Hz of the peak

33
Q

What are labile protons, and what happens to them in the presence of heavy water (D2O)?

A

Labile protons = easily exchangeable (–NH or -OH)

In presence of heavy water, H-1 get exchanged for H-2
H-2 cannot be detected by HSQC because gives out different resonance freq

34
Q

How are hydrogen bonds useful in determining standard secondary structure and tertiary structures in NMR?***

A
35
Q

What 3 restraints are usually used for NMR structure calculation?

A

NOE distance restraints=strong, medium, weak

Dihedral angle restraints from coupling constant

Hydrogen bond restraints

36
Q

What does the computer do to generate an ensemble of structures that obey the constraints?

A

Use simulated annealing and energy minimization

37
Q

What happens if the number of constraints is larger?

A

The ensemble structure will be very similar to each other

Having a SMALL root mean square deviation (rmsd) between their backbone and side chain

38
Q

Summarize the steps of protein NMR

A

Get a solution of proteins with C-13 and N-15

Triple resonance 3D experiments

Backbone and side chain sequential assignment by through-bond experiments

Distance and dihedral angle restraints through-space and coupling constant experiments

39
Q
A
40
Q

What are the cons to NMR?

A

Protein size is limited

High solubility is needed

Conformational exchange problems

Distance restraints assignment and structure refinement

41
Q

What are the prerequisites needed for NMR?

A

Over-expression of folded protein in minimal medium

~95% purity

Highly soluble

Stable at greater than or equal to 25 degrees for a week

Protein Structure & Function (11 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions